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, 12 (3), 449-456

Comparison of the Cardiomyogenic Potency of Human Amniotic Fluid and Bone Marrow Mesenchymal Stem Cells

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Comparison of the Cardiomyogenic Potency of Human Amniotic Fluid and Bone Marrow Mesenchymal Stem Cells

Manali Jain et al. Int J Stem Cells.

Abstract

Background and objectives: Most studies in cardiac regeneration have explored bone marrow mesenchymal stem cells (BM-MSC) with variable therapeutic effects. Amniotic fluid MSC (AF-MSC) having extended self-renewal and multipotent properties may be superior to bone marrow MSC (BM-MSC). However, a comparison of their cardiomyogenic potency has not been studied yet.

Methods: The 5-azacytidine (5-aza) treated AF-MSC and BM-MSC were evaluated for the expression of GATA-4, Nkx2.5 and ISL-1 transcripts and proteins by quantitative RT-PCR and Western blotting, respectively as well as for the expression of cardiomyogenic differentiation markers cardiac troponin-T (cTNT), beta myosin heavy chain (βMHC) and alpha sarcomeric actinin (ASA) by immunocytochemistry.

Results: The AF-MSC as compared to BM-MSC had significantly higher expression of GATA-4 (183.06±29.85 vs. 9.80±0.05; p<0.01), Nkx2.5 (8.3±1.4 vs. 1.82±0.32; p<0.05), and ISL-1 (39.59±4.05 vs. 4.36±0.39; p<0.01) genes as well as GATA-4 (2.01±0.5 vs. 0.6±0.1; p<0.05), NKx2.5 (1.9±0.14 vs. 0.8±0.2; p<0.01) and ISL-1 (1.7±0.3 vs. 0.9±0.1; p<0.05) proteins. The AF-MSC also had significantly elevated expression of cTNT (5.0×104±0.6×104 vs. 3.5×104±0.8×104; p<0.01), β-MHC (15.7×104±0.9×104 vs. 8.2×104±0.6×104; p<0.01) and ASA (18.6×104±4.9×104 vs. 13.1×104±3.0×104; p<0.05) than BM-MSC.

Conclusions: Our data suggest that AF-MSC have greater cardiomyogenic potency than BM-MSC, and thus may be a better source of MSC for therapeutic applications in cardiac regenerative medicine.

Keywords: Amniotic fluid mesenchymal stem cells; Bone marrow mesenchymal stem cells; Cardiac structural markers; Cardiac transcription factors; Cardiomyogenic potency.

Conflict of interest statement

Potential Conflict of Interest

The authors have no conflicting financial interest.

Figures

Fig. 1
Fig. 1
Morphological and characterization of AF-MSC and BM-MSC. (a) Representative photomicrographs (10X, 10 micron) of AF-MSC and BM-MSC showing a trigonal and fibroblastoid morphology respectively in 3rd passage; (b, c) Proliferation rate and Population Doubling Time (PDT) of the both AF-MSC and BM-MSC at different hours; (d) Representative flow cytometric histogram of AF-MSC and BM-MSC showing the presence of MSC markers (CD73, CD90 and CD105) & absence of hematopoietic stem cell markers (CD34, CD45 and HLA-DR). Values expressed as Mean±SD; **p<0.01.
Fig. 2
Fig. 2
Adipogenic, osteogenic and chondrogenic differentiation of AF-MSC and BM-MSC. Representative photomicrographs showing the differentiation of AF-MSC and BM-MSC into (a) adipocytes, as demonstrated with Oil red O Staining and their relative intensity, (b) osteocytes, as demonstrated with Alizarin red staining and their relative intensity, (c) chondrocytes, as demonstrated with Alcian blue staining and their relative intensity. Control AF-MSC and BM-MSC (untreated cells) were negative for Oil red O, Alizarin red and Alcian Blue staining, respectively. Values expressed as Mean±SD; **p<0.01, ***p<0.001, *p<0.05 respectively.
Fig. 3
Fig. 3
Expression of genes and proteins of cardiac transcription factors in AF-MSC and BM-MSC. (a) Representative reverse-transcription polymerase chain reaction photomicrographs showing expression of Cardiac transcription factors in 5-azacytitdine treated and untreated (control) AF-MSC and BM-MSC for GATA-4, NKx2.5 and ISL-1. Values expressed as Mean±SD; **p<0.01, *p<0.05, **p<0.01 respectively. (b) Representative immune-blots showing expression of the GATA-4, NKx2.5 and ISL-1 in both control and 5-aza treated in AF-MSC and BM-MSC (c) their relative intensity applied for comparison of relative protein expression. Values expressed as Mean±SD; *p<0.05, **p<0.01, *p<0.05 between differentiated vs. control respectively.
Fig. 4
Fig. 4
Expression of cardiac structural markers in AF-MSC and BM-MSC. (a) Representative immunofluorescence photomicrographs (40x, 10 micron) of 5 aza treated AF-MSC and BM-MSC staining of cTNT, βMHC, and ASA proteins; (b) their relative intensity was applied for comparison of relative protein expression. Values expressed as Mean±SD; **p<0.01; **p<0.01, *p<0.05 between differentiated vs. control respectively. CTCF (Corrected Total Cell Fluorescence)=Integrated Density−(Area of selected cell×Mean fluorescence of background readings).

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